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Feed and Nutrition
The Effect of Garlic (Allium Sativum) Extract on The Growth of Bacteria Isolated
From Uterus Dairy Cattle
M. Poeloengan 1) and I. Komala 2) 3)
1)
2)
Indonesian Research Center for Veterinary Science, Bogor West Java Indonesia
Faculty of Animal Science Bogor Agricultural University, , West Java Indonesia.
email: [email protected]
3)
Institute of Bioscience, Universiti Putera Malaysia
ABSTRACT
Many dairy farms suffered from tremendous economic losses due to repeat breeding. Laboratory
investigation demonstrated that several bacteria. i. e., Staphylococcus aureus, Streptococcus pyogenes,
Pseudomonas sp., and Klebsiella sp. were responsible for this syndrome. This research was to promote
the uses of traditional medicinal herb as an alternative way to cure repeat breeding. Garlic was as well
known as a herb containing antibacterial effects. The objective of this study was to asses the effects of
garlic extracts on the growth of bacteria isolate which were collected from repeat breeding infected of
local milking cows. A factorial design 3 x 4 was employed for this investigation. Four isolates of
bacteria from repeat breeding, i.e., Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas sp.,
and Klebsiella sp. were used in in-vitro test. Fifteen micro liters each of three concentrations of garlic
extract, i.e., 75, 50, and 25% were dropped in sterile paper disks.The disks were than laid on the MEU
blood media previously inoculated with each of the four isolates and were incubated overnight at 37
degree C. The finding results showed that that the highest concentration of garlic extract inhibited
bacterial growth In further results showed that the garlic extract significantly inhibited the growth of
Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas sp., and Klebsiella sp. [p<.0001]. As
conclusion, the Garlic (Allium sativum) extracts had bactericide effects, especially on Staphylococcus
aureus, and Streptococcus pyogenes; the is positive correlation between the high concentration of garlic extract with
inhibition zones; Staphylococcus aureus shown the most affected by the Garlic extracts at all
concentrations and Garlic extract was able to use effectively for controlling the bacteria growth.
Key words: Garlic, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas sp., Klebsiella sp.
INTRODUCTION
Repeat breeder syndrome is a major source
of economic disadvantage in dairy herds. Cows
that fail to conceive after a defined number of
inseminations with fertile semen (generally 3 or
more) are classified as repeat breeder (Levine,
1999). Around 10%-15% of cows fail to conceive
on time can be classified as repeat breeder
(Hartigan et al., 1972).
Bacterial infection is the most important
among the various causes of the sub fertility
(Dholakia et al., 1987). Such a condition may
cause cervicitis or endometritis of various
degrees, which in turn may lead to embryonic
death and repeat breeding problems (Elliot et al.,
1968). These infections effect fertility by altering
the uterine environment resulting in impairment
of sperm transport, sperm death and hostile
environment to the subsequent development and
maintenance of the conceptualizing to their death.
Faculty of Animal Science, Bogor Agricultural University
Early embryonic death (<42 days) is a major
factor in reproduction failure, which in turn
causes economic loss to the dairy industries
(Rahman et al., 1996).
The Garlic has important efficacy as
antibacterial activity (Ali and Saoji, 2002). The
effects of an aqueous extract of garlic and its
active constituent allicin were tested against 40
drug resistant isolates of the strains of Shigella
dysenteries type 1 and Shigella flexneri,
enterotoxigenic Escherichia coli and Vibrio
cholerae. The aqueous garlic extract and allicin
had potential activities against all of the tested
bacteria, while from the five standards of
antibiotics only gentamcyn was active. Both
allicin and the aqueous extract had a broad
spectrum as antibacterial agents. Allicin appeared
to have the strongest activity compared with that
of the extract and the standard antibiotics (Ahsan,
et al., 1996).
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2009
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Feed and Nutrition
In addition, allicin, one of the sulfurcompounds responsible for garlic's characteristic
odor, is a powerful antibacterial and antiviral
agent that joins forces with vitamin C to help kill
harmful microbes. In research studies, allicin has
been shown to be effective not only against
common infection like colds, flu, stomach
viruses, and Candida yeast, but also against
powerful
pathogenic microbes
including
tuberculosis and botulism (The George Mateljan
Foundation, 2008).
The objective of this study was to asses the
effects of garlic extracts on the growth of bacteria
isolate which were collected from repeat
breeding infected of local milking cows.
MATERIALS AND METHODS
indicated the effectiveness of the garlic extracts
for control the bacterial growth.
Design
This study was designed as a 4 by 3 factorial
experiments. The first factor observed was the
type of bacteria for in vitro test i.e.
Staphylococcus aureus and Streptococcus
pyogenes. The second factor was the
concentration of the Garlic extracts i.e., 75, 50,
and 25%. The parameter observed was the
diameter of inhibition zone of each bacteria. The
analysis of variance was used to analyze the data
of those inhibition zone diameters, and then
Duncan Multiple Range Test (DMRT) procedure
was used to determine furthers differences among
the means of those diameters.
Material
RESULTS AND DISCUSSION
Aqua sterile was used to make three
concentrations of the garlic extract for this
investigation. Then, Mueller Hinton blood agar
and broth media was used as the growth media
for the four bacteria isolates for this study.
Additionally, the blood agar media was also used
as a purification control.
Isolates of Staphylococcus aureus,
Streptococcus pyogenes, Pseudomonas sp., and
Klebsiella sp. were collected from the uterus of
local dairy cows with repeat breeding, raised by
small farmers in Bogor, West Java. These
specimens were later used for bacterial
verification.
The obtained specimens were brought to
BALITVET laboratory at Bogor. Here, they were
cultivated in the blood agar media plates. The
inoculated blood agar plates were incubated for
24 hours at 37 degree C. The bacterial isolates
grown in the blood agar media plates were
identified by employing Cowan and Steel
method’s (1973).
Garlic Extract Procedure
In this procedure, the sterile aqueduct was
added to obtain 75, 50, and 25% of garlic extract
concentrations. Fifteen micro liter from each
concentration was dropped onto sterile paper
disk. Each disk was laid on the MEU blood agar
media after it had been inoculated with four types
of bacteria isolates. Those cultures were
incubated for 24 hours at 37 degree Celsius.
The bacterial growth inhibition zones were
observed and measured. The size of the zones
150
The 1st International Seminar on Animal Industry 2009
1. Effect of Bacteria Isolate Types and
Concentration of Garlic Extract on The
Inhibition Zone
The growth of bacteria was known through
diameter measurement of inhibition zone on the
plate. The finding results of inhibition zones
diameter of bacterial isolates showed at Tabel 1.
Table 1. Diameter of bacterial growth inhibition
zone (mm) as effect of differences level
of garlic extract concentration and
bacteria isolate types/spesies from dairy
uterus
Type of Bacteria
Pseudomonas sp.
Klebsiella sp.
Staphylococcus
aureus
Streptococcus
pyogenes
Average
Concentration
25% 50% 75%
7
9
11
9
11
13
15
18
21
13
15
19
11c
13.25b
16a
Average
9d
11c
18a
15.7b
* Different alphabet code indicated a significant difference at p <
0.05 DMRT
The average of diameter was 9 mm on the
Pseudomonas sp. and 18 mm on the
Staphylococcus aureus. The diameter growth
inhibition zones of Staphylococcus aureus was
greater than Streptococcus pyogenes, and
Klebsiella sp. was greater than Pseudomonas sp.
The analysis of variance showed that the main
effect of type of bacteria, i.e., Staphylococcus
| Faculty of Animal Science, Bogor Agricultural University
Feed and Nutrition
aureus, and Streptococcus pyogenes isolates on
the bacterial growth inhibition zones was highly
significant. Further test, results were presented in
the following Table 1.
Table 1. shown the size of four bacterial
growth inhibition zones at MEU blood agar
media, after being treated with three different
concentration of garlic extracts. In these in vitro
test, Staphylococcus aureus produced the largest
growth
inhibition
zone.,
followed
by
Streptococcus pyogenes. The test also indicated
that the species of Staphylococcus produced
larger growth inhibition than Streptococcus
pyogenes. Statistically, these differences were
significant at alpha equal to or less than 0.01.
Furthermore, Table 2 also show that the
average of the bacterial growth inhibition zones
was getting lower with the lower concentrations
of the garlic extract. Now, what about the main
effect of the garlic concentration increase on the
bacterial growth inhibition zones? As presented
earlier, the Analysis of variance of this effect was
highly significant too. Further test were presented
in Table 2.
Table 2. demonstrated that the first
concentration of the garlic extract, i.e., 75
percent, produced the largest growth inhibition
zone, followed by 50 percent, and 25 percent.
Statistically, the three growth inhibition zones
were different at alpha equal to or less than 0.05.
Therefore, it could be concluded that the higher
concentration of the antibacterial agents in the
garlic extracts make the larger diameter of the
bacterial growth inhibition zones obtained.
Table 2. pointed out that combination of the
garlic extract concentrations and the type of
bacteria produced different growth inhibition
zones of each bacteria studied. Further, Table 3
demonstrated that produced the Staphylococcus
aureus largest growth inhibition zones and
differed significantly with Streptococcus
pyogenes at all concentrations of Garlic Extract.
Table 2. The main effect of the garlic
concentration increase on the bacterial
growth inhibition zones
The Garlic
Extract
25%
50%
75%
Diameter of Growth
Inhibition Zone (mm)
11
13.25
16
Level of
Significance*
c
b
a
* Different alphabet code indicated a significant difference at p <
0.05 DMRT
Faculty of Animal Science, Bogor Agricultural University
2. The Combined Effects of the Garlic Extract
Concentrations and the Type of Bacteria on
the Bacterial Growth Inhibition Zones
As presented earlier, the combined effects of
the garlic extract concentrations and the type of
bacteria on the bacterial growth inhibition zones
were highly significant too. To determine further
differences of the combined effects the Ducan
Multiple Range Tests were performed at all
findings. The result was presented in Table 2.
The explanation to the above finding was
likely found in each species tolerance to surface
tension reducer agents. Generally, bacteria in
nature had a three-layer cell wall structured bond
(Volk and Wheeler, 1988).
This simple structured bond was made of: (1)
cytoplasmic membrane, (2) thicker peptidoglicant
membrane, and (3) varied outer membrane.
According to Volk and Wheeler (1988), the
cytoplasmic membrane was mainly made of
proteins and lipids that were vulnerable to surface
tension reducer agents.
This allicin compound, could destroy the
protein content of the bacterial cell wall. In this
case, the allicine would dissolve the cytoplasmic
membrane and create leaks at the cell wall. In
turn, these leaks would cause losses of important
metabolites
from
the
bacterial
cell.
Staphylococcus aureus and Streptococcus
pyogenes would loose their pathogenic abilities
and died (Volk and Wheeler, 1988). The Garlic
(Allium sativum) extracts had bactericide effects
(garlic extract as agent that destroys bacteria),
especially on Staphylococcus aureus, and
Streptococcus pyogenes.
Secondly, the allicin in the garlic extract
would inactivate a number of the bacterial
enzymatic activities. This too, would deactivate
the Staphylococcus aureus, and Streptococcus
pyogenes abelities and then killed them. Aliicin
can obstruct growth a metabolism of bacteria
with obstruct two enzyme: sistein proteinase and
alkohol dehidrogenase.
CONCLUSIONS
From all of research findings and
interpretation,the conclusion were:
1. The Garlic (Allium sativum) extracts had
bactericide effects, especially on Staphylococcus aureus, and Streptococcus pyogenes.
2. There is positive correlation between the high
concentration of garlic extract with inhibition
zones.
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2009
151
Feed and Nutrition
3. Staphylococcus aureus was shown the most
affected by the Garlic extracts at all
concentrations.
4. Garlic extract was able to use effectively for
controlling the bacteria growth.
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| Faculty of Animal Science, Bogor Agricultural University